LOCUS       sequence-74423-e        6029 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6029)
  AUTHORS   Rhodius VA, Segall-Shapiro TH, Sharon BD, Ghodasara A, Orlova E, 
            Tabakh H, Burkhardt DH, Clancy K, Peterson TC, Gross CA, Voigt CA
  TITLE     Design of orthogonal genetic switches based on a crosstalk map of 
            sigmas, anti-sigmas, and promoters.
  JOURNAL   Mol Syst Biol. 2013 Oct 29;9:702. doi: 10.1038/msb.2013.58.
  PUBMED    24169405
REFERENCE   2  (bases 1 to 6029)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Mol Syst 
            Biol."; date: "2013-10-29"; volume: "9:702. doi"; pages: " 
            10.1038/msb.2013.58"
COMMENT     Sequence Label: pVRa02_2817
FEATURES             Location/Qualifiers
     source          1..6029
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             604..621
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(674..692)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      688..735
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      772..1227
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(859..878)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1069..1090
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1254..1358
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1359..1427,1428..2219)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(1577..1596)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2390..2978
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2879..2898
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3132..3149
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3164..3306
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3232..3251)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3392..3414
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3408..3599)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4408..5490)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=lacI
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     5452..5471
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5491..5568)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5774..5793
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     5877..5896
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5877..5895
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5896..5920
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     RBS             5935..5957
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             5976..5993
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             6003..6026
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
ORIGIN
        1 atgagcgaac agctgaccga tcaggttctg gttgaacgtg ttcagaaagg tgatcagaaa
       61 gcatttaatc tgctggttgt gcgctatcag cataaagttg caagcctggt tagccgttat
      121 gttccgagcg gtgatgttcc ggatgttgtt caagaagcat tcattaaagc atatcgtgcc
      181 ctggatagct ttcgtggtga tagcgccttt tatacctggc tgtatcgtat tgcagtgaat
      241 accgccaaaa attatctggt tgcacagggt cgtcgtccgc ctagcagtga tgttgatgca
      301 attgaagccg aaaactttga aagcggtggt gcactgaaag aaattagcaa tccggaaaat
      361 ctgatgctga gcgaagaact gcgtcagatt gtttttcgta ccattgaaag cctgccggaa
      421 gatctgcgta tggcaattac cctgcgtgaa ctggatggtc tgagctatga agaaattgca
      481 gccattatgg attgtccggt tggcaccgtt cgtagccgta tttttcgtgc acgtgaagcc
      541 attgataata aagttcagcc gctgattcgt cgttaaggat ccaagcttgc ggccgcactc
      601 gagcaccacc accaccacca ctgagatccg gctgctaaca aagcccgaaa ggaagctgag
      661 ttggctgctg ccaccgctga gcaataacta gcataacccc ttggggcctc taaacgggtc
      721 ttgaggggtt ttttgctgaa aggaggaact atatccggat tggcgaatgg gacgcgccct
      781 gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg
      841 ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg
      901 gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac
      961 ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct
     1021 gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt
     1081 tccaaactgg aacaacactc aaccctatct cggtctattc ttttgattta taagggattt
     1141 tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt
     1201 ttaacaaaat attaacgttt acaatttcag gtggcacttt tcggggaaat gtgcgcggaa
     1261 cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac
     1321 cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg
     1381 tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc
     1441 tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg
     1501 atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga
     1561 gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc
     1621 aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag
     1681 aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga
     1741 gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg
     1801 cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga
     1861 atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgcagcaatg gcaacaacgt
     1921 tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact
     1981 ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt
     2041 ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg
     2101 ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta
     2161 tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac
     2221 tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat ttttaattta
     2281 aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt
     2341 tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt
     2401 tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt
     2461 gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc
     2521 agataccaaa tactgtcctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg
     2581 tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg
     2641 ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt
     2701 cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac
     2761 tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg
     2821 acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg
     2881 gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat
     2941 ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt
     3001 tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg
     3061 attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa
     3121 cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcctgatg cggtattttc
     3181 tccttacgca tctgtgcggt atttcacacc gcatatatgg tgcactctca gtacaatctg
     3241 ctctgatgcc gcatagttaa gccagtatac actccgctat cgctacgtga ctgggtcatg
     3301 gctgcgcccc gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg
     3361 gcatccgctt acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca
     3421 ccgtcatcac cgaaacgcgc gaggcagctg cggtaaagct catcagcgtg gtcgtgaagc
     3481 gattcacaga tgtctgcctg ttcatccgcg tccagctcgt tgagtttctc cagaagcgtt
     3541 aatgtctggc ttctgataaa gcgggccatg ttaagggcgg ttttttcctg tttggtcact
     3601 gatgcctccg tgtaaggggg atttctgttc atgggggtaa tgataccgat gaaacgagag
     3661 aggatgctca cgatacgggt tactgatgat gaacatgccc ggttactgga acgttgtgag
     3721 ggtaaacaac tggcggtatg gatgcggcgg gaccagagaa aaatcactca gggtcaatgc
     3781 cagcgcttcg ttaatacaga tgtaggtgtt ccacagggta gccagcagca tcctgcgatg
     3841 cagatccgga acataatggt gcagggcgct gacttccgcg tttccagact ttacgaaaca
     3901 cggaaaccga agaccattca tgttgttgct caggtcgcag acgttttgca gcagcagtcg
     3961 cttcacgttc gctcgcgtat cggtgattca ttctgctaac cagtaaggca accccgccag
     4021 cctagccggg tcctcaacga caggagcacg atcatgcgca cccgtggggc cgccatgccg
     4081 gcgataatgg cctgcttctc gccgaaacgt ttggtggcgg gaccagtgac gaaggcttga
     4141 gcgagggcgt gcaagattcc gaataccgca agcgacaggc cgatcatcgt cgcgctccag
     4201 cgaaagcggt cctcgccgaa aatgacccag agcgctgccg gcacctgtcc tacgagttgc
     4261 atgataaaga agacagtcat aagtgcggcg acgatagtca tgccccgcgc ccaccggaag
     4321 gagctgactg ggttgaaggc tctcaagggc atcggtcgag atcccggtgc ctaatgagtg
     4381 agctaactta cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg
     4441 tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc
     4501 cagggtggtt tttcttttca ccagtgagac gggcaacagc tgattgccct tcaccgcctg
     4561 gccctgagag agttgcagca agcggtccac gctggtttgc cccagcaggc gaaaatcctg
     4621 tttgatggtg gttaacggcg ggatataaca tgagctgtct tcggtatcgt cgtatcccac
     4681 taccgagata tccgcaccaa cgcgcagccc ggactcggta atggcgcgca ttgcgcccag
     4741 cgccatctga tcgttggcaa ccagcatcgc agtgggaacg atgccctcat tcagcatttg
     4801 catggtttgt tgaaaaccgg acatggcact ccagtcgcct tcccgttccg ctatcggctg
     4861 aatttgattg cgagtgagat atttatgcca gccagccaga cgcagacgcg ccgagacaga
     4921 acttaatggg cccgctaaca gcgcgatttg ctggtgaccc aatgcgacca gatgctccac
     4981 gcccagtcgc gtaccgtctt catgggagaa aataatactg ttgatgggtg tctggtcaga
     5041 gacatcaaga aataacgccg gaacattagt gcaggcagct tccacagcaa tggcatcctg
     5101 gtcatccagc ggatagttaa tgatcagccc actgacgcgt tgcgcgagaa gattgtgcac
     5161 cgccgcttta caggcttcga cgccgcttcg ttctaccatc gacaccacca cgctggcacc
     5221 cagttgatcg gcgcgagatt taatcgccgc gacaatttgc gacggcgcgt gcagggccag
     5281 actggaggtg gcaacgccaa tcagcaacga ctgtttgccc gccagttgtt gtgccacgcg
     5341 gttgggaatg taattcagct ccgccatcgc cgcttccact ttttcccgcg ttttcgcaga
     5401 aacgtggctg gcctggttca ccacgcggga aacggtctga taagagacac cggcatactc
     5461 tgcgacatcg tataacgtta ctggtttcac attcaccacc ctgaattgac tctcttccgg
     5521 gcgctatcat gccataccgc gaaaggtttt gcgccattcg atggtgtccg ggatctcgac
     5581 gctctccctt atgcgactcc tgcattagga agcagcccag tagtaggttg aggccgttga
     5641 gcaccgccgc cgcaaggaat ggtgcatgca aggagatggc gcccaacagt cccccggcca
     5701 cggggcctgc caccataccc acgccgaaac aagcgctcat gagcccgaag tggcgagccc
     5761 gatcttcccc atcggtgatg tcggcgatat aggcgccagc aaccgcacct gtggcgccgg
     5821 tgatgccggc cacgatgcgt ccggcgtaga ggatcgagat ctcgatcccg cgaaattaat
     5881 acgactcact ataggggaat tgtgagcgga taacaattcc cctctagaaa taattttgtt
     5941 taactttaag aaggagatat accatgggca gcagccatca tcatcatcat cacagcagcg
     6001 gcctggaagt tctgttccag gggccccat
//
